Agent for use in the case of fructose intolerance

ABSTRACT

5-D-fructose dehydrogenase, optionally in combination with invertase and/or maltase and/or glucose isomerase, may be used to treat fructose intolerance. Other embodiments are also disclosed.

This is a continuation of U.S. Ser. No. 12/093,822, which is a §371application of PCT/IB2006/003223, filed Nov. 15, 2006, and claims thebenefit under 35 U.S.C. §120 of said PCT application, and further claimsthe benefit under 35 U.S.C. §119(e) of U.S. Provisional PatentApplication Ser. No. 60/757,414, filed Jan. 10, 2006, U.S. Ser. No.60/757,424 filed Jan. 10, 2006, U.S. Ser. No. 60/831,050, filed on Jul.17, 2006, and U.S. Ser. No. 60/831,174, filed on Jul. 17, 2006. Thisapplication claims the benefit and/or priority of all theseapplications, as appropriate, and the contents of these applications areincorporated herein by reference.

The present invention refers to an agent for use in the case of fructoseintolerance. In the sense of this patent application, fructoseintolerance does not only mean the medically defined fructoseintolerance and disorders of fructose metabolism (further detailsbelow), but any form of impairment and affliction of health and wellbeing which is caused by the administration of fructose or fructosecontaining foodstuffs or by the release of fructose in the digestivetract of humans or animals from other substances, such as e.g. sucrose.

In the context of this patent application, the terms food and foodstuffare used as synonyms. They mean to also include feed in the sense ofanimal feed. The term “special foodstuff” is defined later on and has aparticular meaning according to the invention.

Fructose is a ketohexose and an important ingredient of food providingenergy. It is present as a component of many di- and oligosaccharides,but also as free fructose, or as both in numerous foodstuffs. Fructoseis contained in food, such as fruits and fruit juices in high amounts,but in particular also in sucrose which is cleaved to fructose andglucose in the organism. In the following, ‘fructose containing’ shouldmean all substances and foodstuffs which either contain fructose in pureform or from which fructose can be released in the digestive tract.

In contrast to glucose, fructose is assimilated into the mucosa cells ofthe small intestine by eased carrier-mediated diffusion. The enzymaticdegradation starts in the liver by the action of the adenosinetriphosphate (ATP) dependent fructokinase, wherein fructose is reactedto fructose 1-phosphate. In the liver and in the kidneys, fructose1-phosphate is cleaved to glycerine aldehyde and dihydroxyacetonephosphate by aldolase B.

Three different disorders of fructose metabolism are known, namely thehereditary fructose intolerance, the intestinal fructose intolerance andthe lack of fructose 1,6-diphosphatase. In addition, fructosuria isknown, which does not need to be treated according to present knowledge.

Hereditary fructose intolerance (HFI) is caused by a lack of aldolase B,an enzyme which is present in the intestinal mucosa, in the liver, inlymphocytes and in the kidneys. Normally, this enzyme degrades overintermediate steps fructose 1-phosphate to fructose 1,6-biphosphate. Inthe case of lack of aldolase B, an accumulation of fructose 1-phosphateresults, and due to inhibition of the degradation of glycogen and thegluconeogenesis severe hypoglycaemias appear, associated with break-outof sweat, tremor, vomiting and cramps after fructose intake. In anunknown manner, acidosis, kidney injuries and aminoaciduria may occur.In babyhood, there is the danger of haemorrhages up to cot death.

Intestinal fructose intolerance exhibits other symptoms. It iswide-spread and occurrs with increasing frequency, in particular in thewestern industrialized nations. Intestinal fructose intolerance iscaused by a disorder of the resorption of fructose as a result of theimpairment of transport processes in the mucosa of the small intestine.The sufferer shows unclear abdominal distress/symptoms and as a resultof the bacterial degradation of the carbohydrates which have beentransferred into the colon increased intestinal production of gas. Theaffliction comprises e.g. bloated feeling, flatulences, colic-likebellyaches, aqueous diarrhoeas and noises in the bowels. Often, a wrongdiagnosis of irritable colon is made.

Lack of fructose 1,6-diphosphatase is a defiency of this key enzyme inthe gluconeogenesis pathway. This deficiency causes an increase of thelevel of lactate in the blood after ingestion of fructose, andhypoglycaemias in the case of an empty stomach with lactacidosis, crampsof muscular hypotonia and coma. Through an adiposis of the liver, alsohepatomegalia may occur.

Not each disorder of fructose metabolism necessarily results in severefructose intolerance. However, even in the case of mild disorders offructose metabolism, impairments of health or well being can often beobserved, which until now could only be influenced by a modification ofthe diet. Also, an excessive intake of fructose-containing food mayresult in impairments of health.

Until now, the symptoms and impairments mentioned above could only beavoided by adhering to a fructose-, sucrose- and sorbitol-free diet.However, maintaining such a diet is very difficult for the sufferers,since fructose is contained in all fruits and many vegetables and isused extensively in the food industry as a sweetener. Also allfoodstuffs which contain e.g. sucrose (household sugar) have to beavoided. It is not only difficult to stick to a corresponding diet—inthe case of hereditary fructose intolerance even a very strict diet—itis also extremely unfavourable in respect to the nutrition physiologyand significantly impairs the quality of life of the sufferers. Thesufferers and the persons skilled in the art (for example physicians,medical specialists, nutrition scientists, nutrition consultants,special journalists, etc.) have assumed for decades that there is noalternative to sticking to a diet. Research aimed at finding analternative to keeping a diet has not been successful. An agent thatwould make it possible to avoid keeping a diet, allowing the intake offructose containing food, would thus meet the urgent need of manysufferers which has existed for decades. A strong prejudice in the artand by the sufferers would be overcome, leading to a dramaticimprovement of the possibilities for the therapy and nutrition in thecase of fructose intolerance, since there is, except for the diet,simply no therapy available. Such an agent would also stop the hithertounsuccessful efforts of persons skilled in the art to enable thesufferers to enjoy a normal diet, including fructose-containing meals,without affliction. The importance of such an agent becomes evident ifone considers the most severe and dangerous consequences for the healthof the sufferers with hereditary fructose intolerance in case they e.g.ingest fructose unknowingly, inadvertently or unintentionally. Thiswould be even more valid for an agent which in addition has no negativeside effects for the health.

Thus, there is provided in accordance with embodiments of the presentinvention an effective agent for use in the case of mild impairments ofthe fructose metabolism as well as in the cases of hereditary andintestinal fructose intolerance and the lack of fructose1,6-diphosphatase, especially in order to allow the intake of fructosecontaining foodstuffs even in the case of fructose intolerance. Further,there is provided in accordance with embodiments of the invention anagent to enable the sufferers of fructose intolerance to eat foods whichthey had to avoid until now due to their fructose content. Furthermore,in accordance with some embodiments of the invention an agent is to beprovided which can reduce or eliminate the occurrence of symptoms offructose intolerance after the intake of fructose.

The subject matter of the invention is an agent which comprises5-D-fructose dehydrogenase (syn. fructose 5-dehydrogenase). The agentaccording to the present invention may facilitate the conversion offructose in the food into 5-keto-D-fructose via dehydrogenation. Thus itis changed in such a manner that it is not available for the bacterialmetabolism, which is characterized by fermentation in the intestine, norcan an accumulation of fructose 1-phosphate in the liver or elsewheretake place. Thus, an increase of the level of lactate in the blood canbe avoided, too. The invention thus provides compositions and methodsthat can be used to enable sufferers of fructose intolerance to ingestfructose-containing foods.

A further subject matter of the present invention is an agent whichreduces the bioavailability of fructose in the human or animal body withthe help of 5-D-fructose dehydrogenase.

Still further, a subject matter of the invention is an agent for use inthe case of impaired fructose metabolism, including fructoseintolerance, which contains a compound effecting the dehydrogenation offructose to 5-keto-D-fructose. This conversion may be achieved e.g.enzymatically by a 5-D-fructose dehydrogenase.

Preferably, the agent according to the invention comprises a5-D-fructose dehydrogenase.

Hence, a subject matter of the invention is in particular an agent foruse in the case of fructose intolerance which comprises a 5-D-fructosedehydrogenase.

Another subject matter of the invention is the use of 5-D-fructosedehydrogenase in the case of a impaired fructose metabolism, includingfructose intolerance.

According to the present invention, a 5-D-fructose dehydrogenase canalso be used for lowering the content of fructose in a foodstuff.

In the context of this application a 5-D-fructose dehydrogenase is anenzyme which can catalyse the dehydrogenation of fructose to5-keto-D-fructose. A possible production method for a 5-D-fructosedehydrogenase is for example described in Ameyama et al., Journal ofBacteriology 1981, 814-823, “D-Fructose Dehydrogenase of Gluconobacterindustrius: Purification, Characterization and Application of EnzymaticMicrodetermination of D-Fructose”, the content of which is incorporatedherein by reference.

In the context of this application special foodstuffs are foodstuffs forparticular nutritional uses, foods for special medical purposes, foodsupplements, dietary supplements, dietetic food supplements, healthfoods, nutraceuticals and food additives. In the context of thisapplication the term foodstuff means to include special foodstuffs asused herein, where applicable.

The invention permits the easy conversion of fructose in a foodstuffinto a form that can avoid the problems associated with fructoseintolerance. Thus, the invention also enables the sufferers of fructoseintolerance to eat foodstuffs which they had to avoid until now due tothe fructose content of these foodstuffs.

According to the present invention, 5-D-fructose dehydrogenase isfurther disclosed for use in medicine, for example as a pharmaceuticalcomposition. Accordingly, the subject matter of the invention includes aproduct which may be 5-D-fructose dehydrogenase or may comprise thisenzyme for the use in a medical treatment. In particular, a method forthe therapeutic treatment of the human or animal body is addressed. Inthe context of this invention, a pharmaceutical composition is aproduct, in particular a substance or substance mixture, suited for usein surgical or therapeutic treatment of the human or animal body and fordiagnostic methods which are performed on the human or animal body. Inthe context of this application pharmaceutical compositions thereforemean to include for example products, in particular substances orsubstance mixtures, which are meant or suitable for curing, alleviating,preventing or detecting fructose intolerance.

The term “treating” when used in connection with the foregoing disordersincludes amelioration, prevention or relief from the symptoms and/oreffects associated with these disorders and includes the prophylacticadministration of an enzyme or a mixture thereof to diminish thelikelihood or seriousness of the condition.

According to a further aspect, the present invention provides afoodstuff which comprises 5-D-fructose dehydrogenase. Still further, thepresent invention provides a foodstuff which contains a sufficientamount of 5-D-fructose dehydrogenase for converting fructose into5-keto-D-fructose. Such a foodstuff may advantageously be produced forexample by a process for treating a foodstuff where the foodstuff isbrought into contact with 5-D-fructose dehydrogenase under suchconditions, that the enzyme is able to convert fructose into5-keto-D-fructose. Such a foodstuff has a reduced fructose contentcompared to the untreated foodstuff and is therefore for the first timesuitable for consumption in the case of fructose intolerance. Afoodstuff can be produced for example by a method where a 5-D-fructosedehydrogenase is added to the foodstuff in such a way that thedehydrogenating effect of the 5-D-fructose dehydrogenase starts onlyafter the intake of the foodstuff. Such a foodstuff containing5-D-fructose dehydrogenase has the same taste as an untreated foodstuff.Therefore, for the first time such foodstuffs are suitable forconsumption in the case of fructose intolerance due to the reduction ofthe fructose content which takes place after the intake.

According to a further aspect of the present invention a medical deviceis provided which comprises 5-D-fructose dehydrogenase. Still further, amedical device is provided which comprises 5-D-fructose dehydrogenase inan amount being effective for the conversion of fructose into5-keto-D-fructose.

The invention will now be described with respect to different aspectsthereof in more detail. Regarding the meaning of the term “agent”, inthe following this is to be understood to include a foodstuff, a specialfoodstuff, a medical device or a pharmaceutical composition.

5-D-Fructose dehydrogenase is a substance which has been known fornearly 40 years without being used for anything but analytical purposes.Accordingly, it was never used in the medical/pharmaceutical field,particularly not for the therapeutic treatment of the human or animalbody or for diagnostic purposes at the human or animal body and veryparticularly not in the case of disorders of the fructose metabolism inhumans or animals. Thus, the present invention addresses the firstmedical indication for 5-D-fructose dehydrogenase.

The agent according to the present invention may be taken orally priorto meals, preferably immediately before meals, together with a meal orimmediately after the meal so that it can exert its dehydrogenatingeffect on fructose in the food pulp. The agent may contain the enzymewithout further additives. However, it is preferable that the agentaccording to the present invention further contains additives which are,for example, pharmaceutically acceptable ingredients and/or ingredientsacceptable for foodstuffs, such as for example extenders, binders,stabilisers, preservatives, flavourings etc. Such additives areconventional and well known for the production of pharmaceuticalcompositions, medical devices, foodstuffs, and special foodstuffs, andthe person skilled in the art knows which additives in which amounts aresuitable for certain dosage forms. Particularly preferably, The agentaccording to the present invention may contain additives, such asdicalcium phosphate, lactose, modified starch, microcrystallinecellulose, maltodextrin and/or fibersol.

It may be advantageous to add to the agent according to the invention anelectron acceptor, for example in an amount of from 1:1 to 1:1000(proportion of acceptor: substrate), preferably 1:2 to 1:200, morepreferred in an amount of from 1:10 to 1:50. Examples of suitableacceptors are, but not limited to, NAD⁺, NADP⁺, FAD⁺, vitamins, such asfor example vitamin C, E or A, ferricyanide, ketones, aldehydes,2,6-di-chloro-phenolindophenol, phenazine methsulfate, nitrobluetetrazolium and mixtures of such acceptors.

The agent according to the present invention can also be added to afoodstuff prior to ingestion. It can even be added to the foodstuff atthe production stage with the purpose of developing its effect afteringestion of the foodstuff. This may be achieved, for example, bymicroencapsulation. With this, the useable fructose content of thefoodstuff may be reduced without altering the taste of the foodstuff ina unfavourable manner. Hence, agents according to the present inventionmay comprise 5-D-fructose dehydrogenase that will be released, or becomeeffective by other means, only in the digestive tract of a human oranimal, in particular in the stomach or the small intestine. Therefore,the invention can be used, for example, in the production of sweets,fruit preparations (e.g. apple puree), jam, honey, chocolate andchocolate products, baked products (e.g. biscuits and cakes), breads,pastas, vegetable dishes, potato dishes, ice cream, cereals, dairyproducts (e.g. fruit yoghurt and pudding), fructose containingbeverages, fructose containing sauces (e.g. tomato ketchup) and fructosecontaining sweeteners. For dishes which are cooked or baked, the agentaccording to the present invention could e.g. be mixed into or sprinkledonto it after cooling down.

Since fructose is used as a sweetener in high amounts in foodstuffs fordiabetics, it is beneficial to add the agent according to the presentinvention to such food before eating or already at the production stage.This allows fructose intolerant diabetics the intake of food fordiabetics, such as the above mentioned foodstuffs in a form suitable fordiabetics (diabetes food).

The agent according to the present invention may also be added to afoodstuff in order to exert its effect on fructose originating fromanother foodstuff after ingestion. The agent can for example be added toa spread so that the reduction of the fructose contained in the breadoccurs after eating the bread with the spread on it without impairingthe taste of the bread. Another example would be spice mixes.

The agent according to the present invention may also be used inimmobilised form. This is particularly useful for the treatment ofliquid foodstuffs. For example, the 5-D-fructose dehydrogenase may beembedded in a matrix which is permeable for fructose: When a fructosecontaining liquid foodstuff flows past the enzyme containing matrix,then fructose is extracted from the foodstuff by the action of theenzyme and converted to 5-keto-D-fructose.

Subject of the present invention are also agents which contain5-D-fructose dehydrogenase in addition to other active ingredients.

The agent may be formulated in any form which is suitable for theintended route of administration. A preferred route of administration isoral administration. For oral administration, the agent may beformulated for example in the form of capsules (coated or non-coated)containing powder, coated or non-coated pellets, granules ormicro-/mini-tablets or in the form of tablets (coated or non-coated)pressed from powder, coated or non-coated pellets, dragees ormicro-/mini-tablets. The agent may also be formulated for example in theform of gel caps or in liquid form as solution, drops, suspension orgel. The agent may also be formulated e.g. as dried or wet moist oralsupplement. The formulation of the agent according to the presentinvention as powder is particularly suitable for admixing withfoodstuff. The powder may be sprinkled onto a meal or mixed into a pulpor beverage. It is particularly beneficial, if the agent offered as bulkpowder is packaged in single dosage amounts, such as in single bags orcapsules, or if it is provided in a dosing dispenser.

For oral administration, 5-D-fructose dehydrogenase may be containedused with acceptable excipients and/or carriers. The term “acceptablecarrier” refers to a carrier for pharmaceutical use, that delivers theactive ingredient to its target site of activity without causingsignificant harm to the treated human or animal. However, the exact formof the carrier is not of substantial importance.

The total content of the carrier within an agent containing 5-D-fructosedehydrogenase is preferably between 5 and 99.9% by weight of thecomposition, more preferably between 10 and 80% even more preferablybetween 25 and 60%.

Suitable excipients and/or carriers include but are not limited tomaltodextrin, calcium carbonate, dicalcium phosphate, tricalciumphosphate, microcrystalline cellulose, dextrose, rice flour, magnesiumstearate, stearic acid, croscarmellose sodium, sodium starch glycolate,crospovidone, sucrose, vegetable gums, lactose, methylcellulose,povidone, carboxymethyl cellulose, corn starch, modified starch,fibersol, gelatine, hydroxypropylmethyl cellulose and the like(including mixtures thereof).

Preferable carriers include calcium carbonate, magnesium stearate,maltodextrin, dicalcium phosphate, modified starch, microcrystallinecellulose, fibersol, gelatine, hydroxypropylmethyl cellulose andmixtures thereof.

The different ingredients and the excipient and/or carrier may be mixedand formed into the desired form using common methods well known to theskilled person. The administration form according to the presentinvention which is suited for the oral route, such as e.g. tablet orcapsule, may be coated with a coating which is resistant against low pHvalues (approximately pH 1 to 2.5) and which dissolves at a pH value ofapproximately 3.0 to 8.0, preferably at a pH value of 3.0 to 6.5 andparticularly preferable at a pH value of 4.0 to 6.0. An optionally usedcoating should be in accordance with the pH optimum of the enzyme usedand its stability at pH values to which the formulation will be exposed.Also a coating may be used which is not resistant to low pH values butwhich delays the release of the enzyme at low pH values. It is alsopossible to prepare the agent according to the present invention ascoated (see above) pellets, granules or micro-/mini-tablets which can befilled into coated or non-coated capsules or which can be pressed intocoated or non-coated tablets. Suitable coatings are, for example,cellulose acetate phthalate, cellulose derivates, shellac,polyvinylpyrrolidone derivates, acrylic acid, polyacrylic acid derivatesand polymethyl methacrylate (PMMA), such as e.g. Eudragit® (from RöhmGmbH, Darmstadt, Germany), in particular Eudragit® L30D-55. The coatingEudragit® L30D-55 is dissolved, for example, at a pH value of 5.5 andhigher. If it is desired to release the enzyme already at a lower pHvalue, this may be achieved e.g. by the addition of sodium hydroxidesolution to the coating agent Eudragit® L30D-55, because in this casecarboxyl groups of the methacrylate would be neutralised. Therefore,this coating will be dissolved, for example, already at a pH value of4.0 provided that 5% of the carboxyl groups are neutralised. Theaddition of about 100 g of 4% sodium hydroxide solution to 1 kg ofEudragit® L30D-55 would result in a neutralisation of about 6% of thecarboxyl groups. Further details about formulation methods andadministration methods can be found in the 21^(st) edition of“Remington: The Science & Practice of Pharmacy”, published 2005 byLippincott, Williams & Wilkins, Baltimore, USA, in the Encyclopedia ofPharmaceutical Technology (Editor James Swarbrick) and in Prof. Bauer“Lehrbuch der Pharmazeutischen Technologie”, 18^(th) edition, published2006 by Wissenschaftliche Verlagsgesellschaft (ISBN 3804-72222-9). Thecontents of these documents are incorporated herein by reference.

Other suitable pharmaceutically acceptable carriers for use in thepresent invention include, but are not restricted to water, mineral oil,ethylene glycol, propylene glycol, lanolin, glyceryl stearate, sorbitanstearate, isopropyl myristate, isopropyl palmitate, acetone, glycerine,phosphatidylcholine, sodium cholate or ethanol.

The compositions for use in the present invention may also comprise atleast one co-emulsifying agent which includes but is not limited tooxyethylenated sorbitan monostearate, fatty alcohols, such as stearylalcohol or cetyl alcohol, or esters of fatty acids and polyols, such asglyceryl stearate.

The agents to be used according to the present invention may be providedin a stabilized form. Generally, stabilisation methods and procedureswhich can be used include any and all methods for the stabilisation ofchemical or biological material which are known in the art and suitedfor the particular purpose, comprising e.g. the addition of chemicalagents, methods which are based on temperature modulation, methods whichare based on irradiation or combinations thereof. Chemical agents thatmay be used according to the present invention include, among others,preservatives, acids, bases, salts, antioxidants, viscosity enhancers,emulsifying agents, gelatinizers, and mixtures thereof.

Usually, the industrial production of enzymes is performed in atechnical fermentation process using suitable microorganisms (bacteria,moulds, fungi). Usually, the strains are recovered from naturalecosystems according to a special screening protocol, isolated as purecultures as well as improved in their properties with respect to theenzyme spectrum and biosynthesis performance (volume/time yield). Enzymeproduction may also be carried out by methods developed in the future.

The 5-D-fructose dehydrogenase is commercially available (e.g.Sigma-Aldrich and Toyobo Enzymes) and is usually prepared in amicrobiological way with the help of the microorganism Gluconobacterindustrius. However, the invention is not limited to the enzymes whichare presently commercially available, but relates generally to enzymeswhich can catalyse the conversion of fructose—specifically or nonspecifically—to 5-keto-D-fructose. A person skilled in the art canprepare suitable further enzymes by methods presently known in the artor by methods which may be developed in the future, for example bymutagenesis of the gene for 5-D-fructose dehydrogenase which is presentin Gluconobacter industrius. The enzyme may also be prepared with thehelp of other microorganisms, such as fungi, in sufficient amounts andthe required purities, also by the use of the genetic engineeringmethods which are presently known or may be developed in the future. Forexample, if it is desired to produce the enzyme with anothermicroorganism, then the genetic information of a microorganism which hasbeen found initially by extensive screening and which has proved to be asuitable source of the enzyme with the desired properties can betransferred to a microorganism which is normally used for the productionof enzymes. Also, the modification of the enzyme itself and theproduction of the enzyme by means of methods which are presently knownor may be developed in the future in the area of industrial enzymedevelopment and enzyme production, such as genetic engineering, ispossible. The use and the manner of performing of all these methods fordeveloping and producing the enzyme with the desired purities andactivities and with the desired properties, in particular with respectto stabilities of the enzyme regarding the pH value, regarding optima ofthe pH value, temperature stabilities and temperature optima, are wellknown to a person skilled in the art. The explanations in chapter 2(page 82 to page 130) of the textbook “Lebensmittel-Biotechnologie andErnährung” of Heinz Ruttloff, Jürgen Proll and Andreas Leuchtenberger,published by Springer Verlag 1997 (ISBN 3-540-61135-5) describe thesemethods in detail. Further information can be found These methods arealso described in “Advances in Fungal Biotechnology for Industry,Agriculture, and Medicine” by Jan S. Tkacz, Lene Langeand (published in2004, ISBN 0-306-47866-8), in “Enzymes in Industry: Production andApplications” by Wolfgang Aehle (Editor), published in 2004, ISBN3527295925 and in “Microbial Enzymes and Biotransformations” byJose-Luis Barredo (Humana Press 2005, ISBN 1588292533). These documentsare herewith incorporated into the patent application by reference. Allthis also applies to the enzymes mentioned below that can optionally beadded to the agent according to the present invention.

The activity of 5-D-fructose dehydrogenase is defined in units (assayavailable e.g. from Sigma-Aldrich), wherein one unit is the amount of5-D-fructose dehydrogenase that converts one micromole of D-fructose to5-keto-D-fructose per minute at pH 4.5 and 37° C. The assay is availablefrom Sigma-Aldrich. Generally, the activity of the 5-D-fructosedehydrogenase per dose unit should be between 10 and 5 million units,preferably between 25 and 2.5 million units and particularly preferablybetween 50 and 1 million units. The wide range of the above mentioneddosages may be explained by the fact that the agent according to thepresent invention is applicable to three different types of fructoseintolerance, namely the hereditary fructose intolerance, the intestinalfructose intolerance and the lack of fructose 1,6-diphosphatase, each indifferent degrees of severity, as well as to mild disorders of thefructose metabolism. Furthermore, the different dosages also result fromthe fact that depending on the specific food intake widely varyingamounts of fructose enter the organism.

The agent according to the present invention may comprise one or moreadditional enzymes, such as invertase (synonymous tobeta-fructofuranosidase or betafructosidase). Invertase can cleavefructose off chemical compounds from the fructose side and may thusrelease fructose from such compounds. It can for example cleave sucrose(household sugar) to glucose and fructose. In another embodiment theagent according to the present invention also comprises the enzymemaltase (syn. alpha-glucosidase) alone or in combination with invertase.This enzyme may also release fructose by cleaving glucose from e.g.sucrose. By the addition of invertase and/or maltase to the agentaccording to the present invention, the endogenis release of fructosefrom fructose containing substances or foodstuffs, in particular fromsucrose, may be both, promoted and accelerated, so that the conversionof fructose to 5-keto-D-fructose which is catalysed by the 5-D-fructosedehydrogenase may occur earlier. Therefore, the addition of invertaseand/or maltase to the agent according to the present invention, may havethe benefit of reducing the required amount of 5-D-fructosedehydrogenase. Such combinations have also never been used in thetherapeutic treatment of the human or animal body or for diagnosticpurposes of the human or animal body.

In another embodiment the agent according to the present invention alsocomprises the enzyme glucose isomerase. A glucose isomerase in thecontext of this application is an enzyme that is able to catalyze aconversion of fructose to glucose. This conversion can also be broughtabout, for example, by a xylose isomerase. Thus, such a xylose isomeraseis, in the sense of this invention, also a glucose isomerase. A possiblemethod for the production of a xylose isomerase is, for example,described in Yamanaka, Biochimica et Biophysika Acta, Volume 151 (3),1968, 670-680, “Purification, Crystallization and Properties of theD-Xylose Isomerase from Lactobacillus brevis” and in Yamanaka, Methodsin Enzymology, Volume 41, 1971, 466-471, “D-Xylose Isomerase fromLactobacillus brevis”.

Glucose isomerase has the property of converting glucose into fructoseand vice versa with an equilibrium concentration of approximately 50%glucose and 50% fructose. Glucose isomerase is therefore ideally suitedfor supporting the action of 5-D-fructose dehydrogenase: Fructose is amonosaccharide which is only slowly absorbed from the intestine into thebloodstream. In contrast Glucose is a monosaccharide which is absorbedquickly from the intestine into the blood stream and which does not posea problem for people with fructose intolerance. If a fructose intolerantperson ingest e.g. apple juice the glucose isomerase will try toestablish the aforementioned equilibrium. Therefore the glucoseisomerase will start to convert fructose into glucose. The glucose thatresults from this conversion process will be absorbed in the intestine.This prevents the achievement of the aforementioned equilibrium.Therefore the glucose isomerase will continue converting fructose toglucose in the food pulp until the aforementioned equilibrium is reachedor until there in no fructose left. Thus, it can be beneficial tocombine 5-D-fructose dehydrogenase not only with invertase and/ormaltase but also with glucose isomerase. In this embodiment, as5-D-fructose dehydrogenase converts fructose to 5-keto-D-fructose, atthe same time the glucose isomerase converts fructose to glucose, whichis also harmless for people with fructose intolerance.

Therefore, the addition of glucose isomerase to the agent according tothe present invention, can reduce the required amount of 5-D-fructosedehydrogenase. The combination of 5-D-fructose dehydrogenase withglucose isomerase and optionally with invertase and/or maltase has alsonever been used in the therapeutic treatment of the human or animal bodyor for diagnostic purposes of the human or animal body.

Invertase and maltase are compounds which have been known for decadesand are commercially available (e.g. BioCat Inc., Troy, USA, NovozymesA/S, Denmark, Sigma Aldrich or Toyobo Enzymes, Japan). So far, invertasehas been used almost exclusively in the production of invert sugar,invert honey and chocolate dishes, such as e.g. chocolate candies. Ithas never been used before in combination with 5-D-fructosedehydrogenase in the medical/pharmaceutical field, and in particular notin the case of disorders of the fructose metabolism in humans oranimals. Until now, this enzyme combination has never been used in thetherapeutic treatment of the human or animal body or for diagnosispurposes. Thus, herewith the first medical indication for thecombination of 5-D-fructose dehydrogenase and invertase is disclosed.The same applies to the combination of 5-D-fructose dehydrogenase withmaltase and to the combination of 5-D-fructose dehydrogenase withinvertase and maltase.

The activity of invertase is measured in Sumner Units (SU, assayavailable e.g. from Bio-Cat Inc., Troy, Va., USA). One SU is defined asthe amount of the enzyme which converts 1 mg of sucrose to glucose andfructose under standard test conditions within 5 minutes at 20° C. and apH value of 4.5. If the agent according to the present invention alsocontains invertase, the activity of the invertase per dose unit shouldbe between 50 and 250,000 SU, preferably between 100 and 150,000 SU andparticularly preferably between 150 and 100,000 SU per dose unit.

The activity of maltase is defined in units, wherein one unit is theamount of maltase which will convert maltose to D-glucose at a rate ofone milligram per minute at 37° C. and a pH of 4.0 in a 10% maltosesolution by weight.

Where the agent according to the present invention also contains maltasethe activity per dose unit should be between 100 and 100,000 units,preferably between 200 and 50,000 units and particularly preferablybetween 500 and 20,000 units.

Glucose isomerase is a compound that has been known for more than 40years and has only been used for starch saccharification to date. In theindustry, it is commonly used in immobilized form for the conversion ofglucose into fructose as well as for the conversion of fructose intoglucose.

Glucose isomerase is commercially available (e.g. Sigma-Aldrich orNovozymes A/S, Denmark) and usually prepared in a microbiological waywith the help of the micro-organism Streptomyces murinus, Where theagent according to the present invention also contains glucoseisomerase, the composition should contain glucose isomerase in an amountof 0.01 to 100,000 GIU, preferably of 0.05 to 10,000 GIU andparticularly preferably of 0.1 to 1,000 GIU per dose unit. One unit ofthis enzyme is defined as a glucose isomerase unit (GIU) that converts 1g of glucose into fructose at a pH value of 6.0 and at a temperature of37° C. from a solution of initially 10% (percent by weight, i.e. 10 g ofglucose +90 g of water) in 5 minutes.

If the agent according to the present invention comprises one or more ofthe aforementioned optional enzymes, then they should be used insufficient amounts—as is the case for the 5-D-fructose dehydrogenase—sothat they can develop a sufficient enzyme activity for the intendedpurpose, e.g. sufficient invertase, so that an amount of sucrose usuallyingested with a normal meal (e.g. 15 g) can be cleaved.

The physiologically present electrolytes will generally be sufficientfor the function of glucose isomerase.

But it may also be advantageous to add electrolytes to the agentaccording to the present invention, preferably in an amount of 0.0001%to 0.1% of the substrate (glucose). Examples of the electrolytesinclude, but are not limited to, MgSO₄, Na₂CO₃, NaHCO₃, NaOH, Na₂SO₄,MgCO₃, H₂SO₄, NaS₂O₃, NaS₂O₅ (including mixtures thereof).

It may also be advantageous to add metal ions, in particular cations,such as Mn²⁺, Mg²⁺, Ca²⁺, Zn²⁺, Fe²⁺, Co²⁺ or Cu²⁺, including mixturesthereof, to the agent according to the present invention, namelypreferably in a molar ratio of 10⁻⁶ to 10⁻². For the above mentioned(xylose) glucose isomerase which is described by Yamanaka, in particularMn²⁺ is a suitable cation.

In the case of intestinal fructose intolerance it is particularlypreferable that the agent according to the present invention maycontain—besides 5-D-fructose dehydrogenase and optionally invertaseand/or maltase and/or glucose isomerase—also glucose in an amount of 50mg to 50,000 mg, preferably 500 mg to 25,000 mg and most preferably1,000 mg to 15,000 mg per dose unit. This is because glucose acceleratesthe resorption of fructose in the intestine.

In the case of hereditary fructose intolerance it is particularlypreferable that the agent according to the present invention maycontain—besides 5-D-fructose dehydrogenase and optionally invertaseand/or maltase and/or glucose isomerase—also folic acid in an amount of1 mg to 100 mg, preferably 2 mg to 50 mg and particularly preferably 3mg to 10 mg per dose unit. This is because folic acid increases theactivity of aldolase B.

In case the agent according to the present invention is added to afoodstuff before ingestion or already at the production stage, theactivity of 5-D-fructose dehydrogenase should be between 10 and 150,000units, preferably between 25 and 100,000 units and particularlypreferably between 50 and 50,000 units per gram fructose in thefoodstuff.

Further, the present invention provides methods for diagnosing fructoseintolerance, for example by administering to a person exhibiting thesymptoms of fructose intolerance a capsule or other formulationcomprising 5-D-fructose dehydrogenase or another suitable enzyme thatconverts fructose to a form that is biologically inactive in humans, orby administering to a person known to exhibit the symptoms of fructoseintolerance both an amount of fructose and a capsule or otherformulation comprising a sufficient amount of 5-D-fructose dehydrogenaseor another suitable enzyme that converts fructose to a form that isbiologically inactive in humans to convert at least a portion of theadministered fructose to a form that is at least one of (a) biologicallyinactive in the human body, (b) not digestible in the human digestivetract and (c) not metabolizable in the human body, and assessing theresults of the administration of the enzyme. In some embodiments, theportion is at least 50% of the administered fructose. In otherembodiments, the portion is at least 75% of the administered fructose.In other embodiments, the portion is at least 90% of the administeredfructose. In other embodiments, the portion is at least 100% of theadministered fructose.

Capsule sizes mentioned below refer to the size definitions used byCapsugel Belgium BVBA, Bornem, Belgium. The size of the capsules shouldbe chosen according to the specific formulation of the agent.

An example for the formulation of the agent according to the presentinvention is the formulation in capsule form with capsules of size 3containing 75 mg of 5-D-fructose dehydrogenase with an activity of 90units/mg and 85 mg of dicalcium phosphate.

Another example for the a dosage form according to the present inventionconsists of capsules of size 1 which contain 150 mg of 5-D-fructosedehydrogenase with an activity of 90 units/mg, 5 mg of folic acid and150 mg of maltodextrin.

A further dosage form according to the present invention may consists ofcapsules (size 3) containing 55 mg of 5-D-fructose dehydrogenase with anactivity of 500 units/mg, 50 mg of invertase with an activity of 200 SUunits/mg and 65 mg of dicalcium phosphate.

A further dosage form according to the present invention consists ofcapsules (size 3) which contain 55 mg of 5-D-fructose dehydrogenase withan activity of 500 units/mg, as well as 50 mg of maltase with anactivity of 200 units/mg and 65 mg of dicalcium phosphate.

A further dosage form according to the present invention consists ofcapsules (size 1) which contain 110 mg of 5-D-fructose dehydrogenasewith an activity of 500 units/mg as well as 100 mg of invertase with anactivity of 200 SU units/mg and 90 mg of maltodextrin.

The invention may for example contain between 10 and 5 million units of5-D-fructose dehydrogenase, between 50 and 250,000 units of invertase,between 100 and 100,000 units of maltase, between 0.01 and 100,000 GIU,between 50 mg and 50 g of glucose and/or between 1 mg and 100 mg offolic acid per dose unit.

Furthermore, suitable additives in useful amounts may be used.

The invention may be provided for medical purposes and non medicalpurposes, e.g. as a pharmaceutical composition, medical device,foodstuffs or special foodstuffs.

In summary, 5-D-fructose dehydrogenase is suited in an excellent mannerfor use in the case of fructose intolerance or any disturbance offructose metabolism. According to preferred embodiments of theinvention, 5-D-fructose dehydrogenase optionally in combination withinvertase and/or maltase and/or glucose isomerase and/or glucose and/orfolic acid are provided as a medicament or are used for the preparationof a medicament for the treatment of fructose intolerance.

Thus, there is provided, in accordance with embodiments of theinvention, a 5-D-fructose dehydrogenase, optionally in combination withinvertase and/or maltase and/or glucose isomerase, for use in medicine,preferably in the form of a pharmaceutical composition. There is alsoprovided, in accordance with embodiments of the invention, apharmaceutical composition comprising 5-D-fructose dehydrogenase,optionally in combination with invertase and/or maltase and/or glucoseisomerase. In some embodiments, the pharmaceutical composition includesone or more enzyme(s) protected by a coating so as to be stable at pHvalues of less than 4, preferably less than 3. In some embodiments, thepharmaceutical composition is in a form for oral administration. In someembodiments, the pharmaceutical composition is in a form suited to beadded to food at the production stage of the same and/or before eating.

There is also provided, in accordance with embodiments of the invention,a medical device comprising 5-D-fructose dehydrogenase, optionally incombination with invertase and/or maltase and/or glucose isomerase. Insome embodiments, the medical device includes one or more enzyme(s)protected by a coating so as to be stable at pH values of less than 4,preferably less than 3. In some embodiments, the medical device is in aform for oral administration. In some embodiments, the medical device isin a form suited to be added to food at the production stage of the sameand/or before eating.

There is also provided, in accordance with embodiments of the invention,a foodstuff comprising 5-D-fructose dehydrogenase in combination withinvertase and/or maltase. In some embodiments, the foodstuff is aspecial foodstuff comprising 5-D-fructose dehydrogenase, optionally incombination with invertase and/or maltase and/or glucose isomerase. Insome embodiments, the foodstuff includes one or more of theabove-mentioned enzymes protected by a coating so as to be stable at pHvalues of less than 4, preferably less than 3. In some embodiments, thefoodstuff is in a form suited to be added to food at the productionstage of the same and/or before eating.

There is also provided, in accordance with embodiments of the invention,the use of 5-D-fructose dehydrogenase alone or optionally in combinationwith invertase and/or maltase and/or glucose isomerase for theproduction of a product, preferably a pharmaceutical composition, foruse in the therapy or diagnosis of disorders of fructose metabolism,including fructose intolerance. There is also provided, in accordancewith embodiments of the invention, the use of 5-D-fructose dehydrogenasealone or optionally in combination with invertase and/or maltase and/orglucose isomerase for the production of a product, preferably for theproduction of a pharmaceutical composition, for lowering thebioavailability of fructose in the human or animal body. There is alsoprovided, in accordance with embodiments of the invention, the use of5-D-fructose dehydrogenase alone or optionally in combination withinvertase and/or maltase and/or glucose isomerase for the production ofa product, preferably for the production of a pharmaceuticalcomposition, for lowering the fructose content in foodstuffs. There isalso provided, in accordance with embodiments of the invention, the useof 5-D-fructose dehydrogenase alone or optionally in combination withinvertase and/or maltase and/or glucose isomerase for the production ofa product, preferably for the production of a pharmaceuticalcomposition, for lowering the amount of fructose available to the humanor animal body and/or to bacteria of the intestine of the human oranimal body. In some embodiments, the product comprises anypharmaceutical composition, a medical device, a foodstuff or a specialfoodstuff. In some embodiments, the product is coated in such a mannerthat one or more of the above-mentioned enzymes are protected against pHvalues of less than 4, preferably less than 3. In some embodiments, theproduct is in a form for oral use. In some embodiments, the product issuited to be added to food at the production stage of the same and/orbefore eating. In some embodiments, the product is in a form for use inimmobilised form.

There is also provided, in accordance with embodiments of the invention,a process for the treatment of a foodstuff, the process comprising thesteps of contacting the foodstuff with a 5-D-fructose dehydrogenase,optionally in combination with invertase and/or maltase, and initiatingthe reaction of fructose to 5-keto-D-fructose. There is also provided,in accordance with embodiments of the invention, a process for thetreatment of a foodstuff, comprising the steps of contacting thefoodstuff with a 5-D-fructose dehydrogenase, optionally in combinationwith invertase and/or maltase and/or glucose isomerase, and initiatingthe reaction of fructose to 5-keto-D-fructose and optionally thereaction of fructose to glucose. In some embodiments, as a further stepprior to the reaction ingestion of the foodstuff takes place.

There is also provided, in accordance with embodiments of the invention,a human-ingestible composition of matter which comprises an enzyme thatconverts D-fructose into a form that is biologically inactive in thehuman body or a mixture of such enzymes. There is also provided, inaccordance with embodiments of the invention, a human-ingestiblecomposition of matter which comprises an enzyme that converts D-fructoseinto a form that cannot be digested in the human digestive tract. Thereis also provided, in accordance with embodiments of the invention, ahuman-ingestible composition of matter which comprises an enzyme thatconverts D-fructose into a form that is not metabolizable in the humanbody. In some embodiments, the form is 5-keto-D-fructose. In someembodiments, the enzyme is 5-D-fructose dehydrogenase. In someembodiments, the composition of matter is a dietary supplement or apharmaceutical composition. In some embodiments, the composition ofmatter is a special foodstuff. In some embodiments, the composition ofmatter further comprises at least one pharmaceutically or dietarilyacceptable carrier or excipient. In some embodiments, the composition ofmatter contains the enzyme in microencapsulated form. In someembodiments, the composition of matter is in the form of a capsule ortablet. In some embodiments, the composition of matter is in the form ofgranules or pellets. In some embodiments, the composition of matter isin the form of a solution. In some embodiments, the composition ofmatter is in the form of a gel or suspension. In some embodiments, thecomposition of matter is in the form of a gelcap.

There is also provided, in accordance with embodiments of the invention,a composition of matter which is microencapsulated 5-D-fructosedehydrogenase. There is also provided, in accordance with embodiments ofthe invention, a composition of matter comprising 5-D-fructosedehydrogenase admixed with a human-ingestible substance. In someembodiments, the human-ingestible substance is a pharmaceutically ordietarily acceptable carrier or excipient. In some embodiments, the5-D-fructose dehydrogenase is microencapsulated.

In accordance with some embodiments, the enzyme constitutes between 5and 99.9% by weight of the composition of matter described above. Insome embodiments, the enzyme constitutes between 10 and 80% by weight ofthe composition of matter. In some embodiments, the enzyme constitutesbetween 25 and 60% by weight of the composition of matter.

In accordance with some embodiments, the composition of matter asdescribed above is in unit dosage form and the unit dosage containsbetween 10 and 5 million units of 5-D-fructose dehydrogenase activity.In some embodiments, the unit dosage contains between 25 and 2.5 millionunits of 5-D-fructose dehydrogenase activity. In some embodiments, theunit dosage contains between 50 and 1 million units of 5-D-fructosedehydrogenase activity.

In accordance with some embodiments, the composition of matter asdescribed above comprises a coating which dissolves in an aqueous mediumat a pH of between 3.0 and 8.0. In some embodiments, the coating doesnot dissolve in an aqueous medium at a pH below 3.0. In someembodiments, the coating does not dissolve in an aqueous medium at a pHbelow 4.0. In some embodiments, the coating does not dissolve in anaqueous medium at a pH above 6.5. In some embodiments, the coating doesnot dissolve in an aqueous medium at a pH above 6.0.

In accordance with some embodiments, the composition of matter asdescribed above is a slow-release or extended-release formulation. Insome embodiments, the slow-release or extended-release formulationcomprises a slow-release or extended-release coating.

In accordance with some embodiments, the composition of matter asdescribed above further comprises a second enzyme. In some embodiments,the second enzyme is capable of cleaving fructose from a more complexsugar. In some embodiments, the second enzyme is invertase or maltase.In some embodiments, the second enzyme is invertase, the composition ofmatter is in unit dosage form, and each unit dosage contains between 50and 250,000 Sumner units of invertase activity. In some embodiments,each unit dosage contains between 100 and 150,000 Sumner units ofinvertase activity. In some embodiments, each unit dosage containsbetween 150 and 100,000 Sumner units of invertase activity. In someembodiments, the second enzyme is maltase, the composition of matter isin unit dosage form, and each unit dosage contains between 100 and100,000 units of maltase activity. In some embodiments, each unit dosagecontains between 200 and 50,000 units of maltase activity. In someembodiments, each unit dosage contains between 500 and 20,000 units ofmaltase activity. In some embodiments, the composition of mattercomprises both invertase and maltase.

In accordance with some embodiments, the composition of matter asdescribed above further comprises an additional enzyme that convertsD-fructose into a molecule that is absorbed more quickly than D-fructosefrom the intestine into the bloodstream. In some embodiments, themolecule is D-glucose In some embodiments, the additional enzyme is aglucose isomerase. In some embodiments, the composition of matter is inunit dosage form and each dosage unit contains 0.01 to 100,000 units ofglucose isomerase activity per dosage unit. In some embodiments, eachdosage unit contains 0.05 to 10,000 units of glucose isomerase activityper dose unit. In some embodiments, each dosage unit contains 0.1 to1,000 units of glucose isomerase activity per dose unit. In someembodiments, the glucose isomerase is a xylose isomerase. In someembodiments, the composition of matter further comprises at least one ofan electrolyte and a metal ion. In some embodiments, the electrolyte isselected from the group consisting of MgSO₄, Na₂CO₃, NaHCO₃, NaOH,Na₂SO₄, MgCO₃, H₂SO₄, NaS₂O₃, NaS₂O₅ and mixtures of any of thesecompounds. In some embodiments, the metal ion is selected from the groupconsisting of Mn²⁺, Mg²⁺, Ca²⁺, Zn²⁺, Fe²⁺, Co²⁺, Cu²⁺ and mixtures ofany of these compounds.

In accordance with some embodiments, the composition of matter asdescribed above is in unit dosage form, and each dosage unit furthercomprises from 50 to 50,000 mg of glucose. In some embodiments, eachdosage unit comprises from 500 to 25,000 mg of glucose. In someembodiments, each dosage unit comprises from 1,000 to 15,000 mg ofglucose.

In accordance with some embodiments, the composition of matter asdescribed above is in unit dosage form, and each dosage unit furthercomprises from 1 to 100 mg of folic acid. In some embodiments, eachdosage unit comprises from 2 to 50 mg of folic acid. In someembodiments, each dosage unit comprises from 3 to 10 mg of folic acid.

In accordance with some embodiments, the composition of matter asdescribed above is a foodstuff, and the enzyme is in active form. Insome embodiments, the amount or concentration of the enzyme in thefoodstuff is greater than the naturally occurring concentration oramount of the enzyme in the foodstuff. In some embodiments, thefoodstuff is a fructose-containing foodstuff. In some embodiments, thefoodstuff is a foodstuff which has been baked. In some embodiments, thefoodstuff is a foodstuff which has been cooked. In some embodiments, thefoodstuff is a liquid, paste or broth. In some embodiments, the enzymeis present in microencapsulated form. In some embodiments, the enzyme is5-D-fructose dehydrogenase. In some embodiments, the foodstuff furthercontains a second enzyme in active form. In some embodiments, theconcentration or amount of the second enzyme in the foodstuff is greaterthan the naturally occurring concentration or amount of the secondenzyme in the foodstuff. In some embodiments, the second enzyme isinvertase. In some embodiments, the second enzyme is maltase. In someembodiments, the second enzyme is a glucose isomerase. In someembodiments, the glucose isomerase is a xylose isomerase. In someembodiments, the composition further comprises at least one of anelectrolyte and a metal ion. In some embodiments, the electrolyte isselected from the group consisting of MnSO₄, Na₂CO₃, NaHCO₃, NaOH,Na₂SO₄, MgCO₃, H₂SO₄, NaS₂O₃, NaS₂O₅ and mixtures thereof. In someembodiments, the metal ion is selected from the group consisting ofMn²⁺, Mg²⁺, Ca²⁺, Zn²⁺, Fe²⁺, CO²⁺, Cu²⁺ and mixtures thereof. In someembodiments, the second enzyme is a mixture of at least two of the groupof invertase, maltase and a glucose isomerase. In some embodiments, thesecond enzyme is in microencapsulated form.

In accordance with some embodiments, the composition of matter asdescribed above comprises a mixture of enzymes that convert D-fructoseinto a form that is at least one of (a) biologically inactive in thehuman body, (b) not digestible in the human digestive tract and (c) notmetabolizable in the human body.

In accordance with some embodiments, the foodstuff described above isnot a dough.

In accordance with some embodiments, the enzyme that converts D-fructoseinto a form that is at least one of (a) biologically inactive in thehuman body, (b) not digestible in the human digestive tract and (c) notmetabolizable in the human body is not contained in an inorganic-basedsol-gel biocompatible matrix.

In accordance with some embodiments, the composition is substantiallyfree of substances which are not approved for oral human ingestion.

In accordance with some embodiments, the composition of matter isadapted for oral ingestion.

In accordance with some embodiments, the composition comprises anelectron acceptor. In some embodiments, the electron acceptor isselected from the group consisting of Nicotinamide AdenineDinucleotide+(NAD+), nicotinamide adenine dinucleotidephosphate+(NADP+), flavin adenine dinucleotide+, vitamin C, E or A,ferricyanide, ketones, aldehydes, 2,6-di-chloro-phenolindophenol,phenazine methosulfate and mixtures thereof. In some embodiments, themolar ratio of electron acceptor to fructose is from 1:1 to 1:1000. Insome embodiments, the molar ratio of electron acceptor to fructose isfrom 1:2 to 1:200. In some embodiments, the molar ratio of electronacceptor to fructose is from 1:10 to 1:50.

There is also provided, in accordance with embodiments of the invention,a method of treating fructose intolerance or impaired fructosemetabolism, comprising administering to a human or animal subject anefficacious amount of an enzyme or a mixture of enzymes that convertsD-fructose into a form that is at least one of (a) biologically inactivein the subject body, (b) not digestible in the subject digestive tractand (c) not metabolizable in the subject body. In some embodiments, thesubject is a human subject. In some embodiments, the administeringcomprises administering a human-ingestible composition of matter asdescribed above. In some embodiments, the fructose intolerance ishereditary fructose intolerance. In some embodiments, the fructoseintolerance is intestinal fructose intolerance. In some embodiments, thefructose intolerance is due to a lack of fructose 1,6-diphosphatase. Insome embodiments, the composition of matter is administered immediatelyprior to eating. In some embodiments, the composition of matter isadministered concurrently with a meal. In some embodiments, thecomposition of matter is administered immediately after eating. In someembodiments, the form is 5-keto-D-fructose.

There is also provided, in accordance with embodiments of the invention,a method for diagnosing fructose intolerance, comprising administeringto a human or animal subject exhibiting the symptoms of fructoseintolerance a composition of matter as described above and observing ifthe administering partly or fully ameliorates said symptoms. In someembodiments, the subject is a human subject.

There is also provided, in accordance with embodiments of the invention,a method for diagnosing fructose intolerance, comprising administeringto a human or animal subject known to exhibit the symptoms of fructoseintolerance both (a) an amount of fructose effective to induce suchsymptoms and (b) a composition of matter as described above whichcomprises an enzyme that converts fructose, or mixture of such enzymes,in an amount which is sufficient to convert at least 50% of the amountof fructose, and assessing the results of the administration of theenzyme or mixture thereof. In some embodiments, the compositioncomprises the enzyme that converts fructose, or mixture of such enzymes,in an amount which is sufficient to convert at least 75% of the amountof fructose. In some embodiments, the composition comprises the enzymethat converts fructose, or mixture of such enzymes, in an amount whichis sufficient to convert at least 90% of the amount of fructose. In someembodiments, the composition comprises the enzyme that convertsfructose, or mixture of such enzymes, in an amount which is sufficientto convert substantially all of the amount of fructose. In someembodiments, the enzyme or mixture of enzymes converts D-fructose to5-keto-D-fructose. In some embodiments, the subject is a human subject.

There is also provided, in accordance with embodiments of the invention,a kit comprising an enzyme that converts D-fructose into a form that isat least one of (a) biologically inactive in the human body, (b) notdigestible in the human digestive tract and (c) not metabolizable in thehuman body, or a mixture of such enzymes, and instructions explaininghow to use the enzyme or mixture thereof to diagnose or treat fructoseintolerance. In some embodiments, the enzyme or mixture thereof ispresent as a composition of matter as described above. In someembodiments, the enzyme or mixture of enzymes converts D-fructose into5-keto-D-fructose.

There is also provided, in accordance with embodiments of the invention,a reduced-fructose foodstuff.

There is also provided, in accordance with embodiments of the invention,a method for preparing a reduced-fructose foodstuff, comprisingcontacting a foodstuff or foodstuff precursor with an enzyme thatconverts D-fructose into a form that is at least one of (a) biologicallyinactive in the human body, (b) not digestible in the human digestivetract and (c) not metabolizable in the human body, and completing anyadditional steps necessary to prepare the foodstuff. In someembodiments, the enzyme is 5-D-fructose dehydrogenase. In someembodiments, the foodstuff is not a baked foodstuff. In someembodiments, the foodstuff is not bread. In some embodiments, thefoodstuff is not a dough. In some embodiments, the enzyme convertsD-fructose to 5-keto-D-fructose.

The invention claimed is:
 1. A mammalian ingestible composition ofmatter which is adapted for oral administration selected from apharmaceutical composition and a dietary supplement, said composition ofmatter being in unit dosage form, said composition of matter comprisingisolated, dry, active 5-D-fructose dehydrogenase and a carrier orexcipient that is acceptable for use in pharmaceutical compositions orfoodstuffs, wherein said 5-D-fructose dehydrogenase is not contained inan inorganic-based sol-gel biocompatible matrix, said 5-D-fructosedehydrogenase constitutes between 25 and 80% by weight of thecomposition of matter, and the unit dosage form is a tablet.
 2. Acomposition of matter according to claim 1, further comprising a secondenzyme which is selected from the group consisting of invertase, maltaseand combinations thereof.
 3. A mammalian ingestible composition ofmatter according to claim 1 wherein the composition of matter is adietary supplement.
 4. A mammalian ingestible composition of matteraccording to claim 1 wherein said unit dosage form contains between 10and 5 million units of 5-D-fructose dehydrogenase activity.
 5. Amammalian ingestible composition of matter according to claim 4 whereinsaid unit dosage form contains between 50 and 1 million units of5-D-fructose dehydrogenase activity.
 6. A composition of matteraccording to claim 1 wherein said 5-D-fructose dehydrogenase isprotected by a coating which is stable at pH below
 4. 7. A compositionof matter according to claim 6 wherein said composition of matter is inunit dosage form and said coating protects the entire dosage unit.
 8. Acomposition of matter according to claim 1 wherein said composition ofmatter comprises a coating which dissolves at a pH of 5.5 or higher. 9.A composition of matter according to claim 1 wherein said 5-D-fructosedehydrogenase is not contained in an inorganic-based sol-gelbiocompatible matrix.
 10. A composition of matter according to claims 1wherein said composition of matter comprises a coating which dissolvesin an aqueous medium at a pH of between 3.0 and 8.0.
 11. A compositionof matter according to claim 10 wherein said coating does not dissolvein an aqueous medium at a pH of below 3.0.
 12. A composition of matteraccording to claim 10 wherein said coating does not dissolve in anaqueous medium at a pH below 4.0.
 13. A composition of matter accordingto claims 10 wherein said coating does not dissolve in an aqueous mediumat a pH above 6.5.
 14. A composition of matter according to claim 10wherein said coating does not dissolve in an aqueous medium at a pHabove 6.0.
 15. A composition of matter according to claim 1 wherein saidcomposition of matter is a slow-release or extended-release formulation.16. A composition of matter according to claim 15 wherein saidslow-release or extended-release formulation comprises a slow-release orextended-release coating.